Recent Advances in the Elucidation of Frataxin Biochemical Function Open Novel Perspectives for the Treatment of Friedreich's Ataxia

Abstract

Friedreich's ataxia (FRDA) is the most prevalent autosomic recessive ataxia and is associated with a severe cardiac hypertrophy and less frequently diabetes. It is caused by mutations in the gene encoding frataxin (FXN), a small mitochondrial protein. The primary consequence is a defective expression of FXN, with basal protein levels decreased by 70-98%, which foremost affects the cerebellum, dorsal root ganglia, heart and liver. FXN is a mitochondrial protein involved in iron metabolism but its exact function has remained elusive and highly debated since its discovery. At the cellular level, FRDA is characterized by a general deficit in the biosynthesis of iron-sulfur (Fe-S) clusters and heme, iron accumulation and deposition in mitochondria, and sensitivity to oxidative stress. Based on these phenotypes and the proposed ability of FXN to bind iron, a role as an iron storage protein providing iron for Fe-S cluster and heme biosynthesis was initially proposed. However, this model was challenged by several other studies and it is now widely accepted that FXN functions primarily in Fe-S cluster biosynthesis, with iron accumulation, heme deficiency and oxidative stress sensitivity appearing later on as secondary defects. Nonetheless, the biochemical function of FXN in Fe-S cluster biosynthesis is still debated. Several roles have been proposed for FXN: iron chaperone, gate-keeper of detrimental Fe-S cluster biosynthesis, sulfide production stimulator and sulfur transfer accelerator. A picture is now emerging which points toward a unique function of FXN as an accelerator of a key step of sulfur transfer between two components of the Fe-S cluster biosynthetic complex. These findings should foster the development of new strategies for the treatment of FRDA. We will review here the latest discoveries on the biochemical function of frataxin and the implication for a potential therapeutic treatment of FRDA.

Keywords: Friedreich’s ataxia; frataxin; iron-sulfur cluster; persulfide; therapy.

FIGURE 1

Structure of human frataxin (FXN) mapping conserved amino acids. Structure of human FXN (PDB code 1EKG) highlighting conserved amino acids from alignment of 50 eukaryotic and 50 prokaryotic frataxins (Supplementary Figure 1). Below is the sequence of full length human frataxin. Amino acids colored in dark red have identity/homology percentage between 100 and 60% and those in light red between 60 and 30%.

FIGURE 2

Iron-sulfur (Fe-S) cluster biogenesis pathway. (A) Fe-S cluster biosynthesis is initiated in mitochondria by the ISC machinery that encompasses the scaffold protein ISCU on which [2Fe2S] clusters are assembled, the cysteine-desulfurase NFS1 that provides sulfur in the form of a cysteine-bound persulfide and FDX2/FDXR that reduces the persulfide to sulfide. Frataxin accelerates the formation of the [2Fe2S] cluster on ISCU. The [2Fe2S] cluster is transferred onto GLRX5 with assistance from the ATP-dependent chaperones HSC20 and HSPA9, and then to recipient apo-proteins. Conversion to a [4Fe4S] cluster from the [2Fe2S] cluster is achieved by the ISCA proteins. The ISC machinery generates a sulfur-containing compound (compound X) that is exported by the ABCB7 transporter to the CIA machinery. [4Fe4S] clusters are assembled by the CIA machinery and delivered to their cytoplasmic and nuclear protein acceptors. (B) Mechanism of Fe-S cluster biosynthesis by the ISC machinery: (1) a ferrous iron is inserted into the assembly site of ISCU, (2) two Fe-ISCU form a heterodimer with a dimer of NFS1 (ACP and ISD11 that bind NFS1 are omitted for clarity), (3) NFS1 catalyzes the formation of persulfide on its catalytic cysteine, (4) the persulfide of NFS1 is transferred to ISCU and FXN accelerates this reaction, (5) FDX2/FDXR reduces the persulfide into sulfide, leading to formation of a [2Fe2S] cluster most likely by dimerization of ISCU, (6) the [2Fe2S] cluster carried by ISCU is transferred to acceptor proteins. Step 1–6 describe the FDX2-based reaction. A non-physiological process called the thiol-based reaction allows formation of Fe-S cluster in vitro in the absence of FDX2/FDXR via the following steps: (7) thiols (RSH) such as DTT, L-cysteine or GSH reduces the persulfide of NFS1, which leads to formation of persulfidated thiols (RSSH). FXN accelerates this persulfide cleavage by thiols. (8) the persulfide of NFS1 is regenerated by reaction with L-cysteine, (9) RSSH reacts with a second thiol to form free sulfide in the form of H₂S alongside oxidized RSSR, (10) free sulfide slowly incorporates into Fe-ISCU to form [2Fe2S] and [4Fe4S] clusters by an unknown mechanism, with DTT favoring formation [4Fe4S] clusters.

FIGURE 3

Mapping iron-binders amino acids of frataxin (FXN) within the NFS1-ISD11-ACP-Zn-ISCU-FXN complex. Structure of the human NFS1-ISD11-ACP-Zn-ISCU-FXN complex (PDB code 6NZU) highlighting the amino acids of FXN identified as iron-binders. The amino acids interacting with NFS1 (E96, E100, E108, E111, D115, E121, and D124) or important for the internal structure of FXN (E92, E101) are colored in pink and those not interacting with other amino acids (D104 and D112) are in cyan. ISCU is colored in salmon, the two subunits of the NFS1 dimer are in light green (NFS1) and dark green (NFS1’), FXN is in blue.

FIGURE 4

Structural rearrangement at the zinc site of ISCU upon binding of NFS1 and frataxin (FXN). Zinc site of panel (A) mouse ISCU (PDB code 1WFZ), (B) human NFS1-ISD11-ACP-ISCU complex (PDB code 5WLW) and (C) human NFS1-ISD11-ACP-ISCU-FXN complex (PDB code 6NZU). (D) FXN variants with impaired activity mapped on the structure of the human NFS1-ISD11-ACP-ISCU-FXN complex (PDB code 6NZU). The amino acids in red are involved in the interaction with ISCU, amino acids in purple are involved in interaction with NFS1. ISCU is colored in salmon, the two subunits of the NFS1 dimer are in light green (NFS1) and dark green (NFS1’), FXN is in blue.